Proceedings of the Fourth Genoa Meeting on Hypertension, Diabetes, and Renal Diseases
Glomerulotubular Junction Abnormalities Are Associated with Proteinuria in Type 1 Diabetes
Behzad Najafian*,,
John T. Crosson,
Youngki Kim* and
Michael Mauer*
* Division of Pediatric Nephrology, Department of Pediatrics, and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota
Address correspondence to: Dr. Michael Mauer, MMC 491, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: 612-626-2922; Fax: 612-626-2791; E-mail: mauer002{at}tc.umn.edu
Glomerulotubular junction abnormalities, frequent in proteinuricpatients with type 1 diabetes, may contribute to the progressiveGFR loss in overt diabetic nephropathy. Glomerulotubular junctionabnormalities were examined in patients who have type 1 diabeteswith a wide range of albumin excretion rates (AER). Renal biopsiesfrom five normoalbuminuric patients, five microalbuminuric patients,six proteinuric patients, and five control subjects were studiedby light and electron microscopy. Light microscopy specimenswere serially sectioned to find and classify glomerulotubularjunctions. Glomerular structural parameters were estimated usingstereologic methods. Glomerulotubular junction abnormalitieswere found in 2% of glomeruli from control and normoalbuminuricpatients and in 4% of glomeruli from microalbuminuric patients.In contrast, 71% of glomeruli from proteinuric patients hadglomerulotubular junction abnormalities, including five (8%)atubular glomeruli. Electron microscopy findings were typicalof diabetic nephropathy. Piece-wise linear regression modelswith glomerular, glomerulotubular junction, and interstitialparameters as independent variables provided greater GFR (92%;P < 0.005) and AER (95%; P < 0.01) prediction than multipleregression models (81% for GFR and 72% for AER). Thus, glomerularadhesions and glomerulotubular junction abnormalities help toexplain the progressive GFR loss that is associated with onsetof proteinuria in type 1 diabetes. Moreover, nonlinear modelsprovide better fit for structural–functional relationshipsin patients with type 1 diabetes.
Diabetic nephropathy (DN) remains the leading cause of ESRDin the United States (1). Albumin excretion rate (AER) is thebest predictor of nephropathy risk in patients with diabetes(2). Studies of structural–functional relationships haveimproved the understanding of the pathophysiology of DN. However,much of the variability in renal function observed in patientswith type 1 diabetes is not explained by present models. Mesangialfractional volume [Vv(Mes/glom)] and glomerular basement membrane(GBM) width explained 59% of the variability in AER in a groupof 125 patients who had type 1 diabetes with wide ranges ofrenal structure and function and underwent renal research biopsies.Surface density of peripheral GBM [Sv(PGBM/glom)], AER, andgender, together, explained only 33% of the variability in GFRamong these patients (3). In fact, whereas GFR is normal orincreased in most normoalbuminuric (NA) and microalbuminuric(MA) patients with type 1 diabetes and decreased in most proteinuricpatients, there is substantial overlap in these structural parametersamong these three groups (3). These findings suggest that otherstructural parameters also may be involved in the process ofDN development and progression. Recently, we showed that bothatubular glomeruli (AG) and glomerulotubular junction (GTJ)abnormalities (GTJA), defined as atrophic proximal tubule atthe GTJ, are frequent in proteinuric patients with type 1 diabetes(4). These abnormalities were closely associated with tip lesions(glomerular tuft to Bowman’s capsule adhesions [TBCA]at GTJ area), a finding reported in a variety of renal diseaseswith clinical proteinuria (5–7). This study also foundthat, along with the classical diabetic glomerulopathy lesions,these GTJA, together with tubulointerstitial parameters, predictedmost of the variability in GFR among these proteinuric patients(4). However, because NA and MA patients were not included,the relationship of GTJA and AG to AER could not be assessedin this study. Our study explores the frequency of tip lesions,GTJA, and AG over a wide range of AER; investigates the frequencyof TBCA in these patients; and presents improved statisticalmodels for the understanding of structural–functionalrelationships in patients with type 1 diabetes.
Patients
Five NA, five MA, and six proteinuric patients with type 1 diabetesand five normal kidney donors (controls) were included in thisstudy. The groups were matched for age, gender, and diabetesage at onset and duration in groups with type 1 diabetes (Table 1).Some of the data from three of the proteinuric patients withtype 1 diabetes and one of the control subjects were publishedpreviously (4). All studies were performed with permission ofthe Committee on the Use of Human Subjects in Research at theUniversity of Minnesota and after informed consent was obtained.
Table 1. Demographic and clinical data of patients and control subjectsa
Clinical Studies
Patients with type 1 diabetes were admitted to the General ClinicalResearch Center (GCRC) at the University of Minnesota for renalfunctional studies and research renal biopsies. AER was measuredas described previously (8). Patients were classified into threegroups on the basis of AER levels in at least two of three consecutiveurine samples: NA, AER <20 µg/min; MA, AER 20 to 200µg/min; and proteinuric, AER >200 µg/min. GFRwas estimated from the clearance of iothalamate or iohexol,both shown to be interchangeable with the clearance of inulin(9). Values of GFR were not corrected for body surface areain the exploratory models (10). Hemoglobin A1c (HbA1c) was measuredby HPLC. BP was taken as the mean value of multiple measurementsthat were done by GCRC nurses using automated equipment. Hypertensionwas defined as BP >130/80 mmHg (11) or the use of antihypertensivedrugs.
Light Microscopy Studies.
Zenker-fixed percutaneous renal biopsy tissues were embeddedin paraffin, serially sectioned at 5-µm thickness, andstained with periodic acid-Schiff. Eight to 13 complete nonsclerosedglomeruli with intact Bowman’s capsules that were observablethrough serial sections were acquired using the disector method(12), which allows unbiased sampling of glomeruli regardlessof their size. Glomeruli with sclerosis of >75% of the tuftson serial sections were enumerated for structural–functionalrelationship models but not considered for stereologic measurements.Glomerular tuft and corpuscle (tuft plus Bowman’s space)volumes were measured using the Cavalieri principle on digitalimages of serial profiles of glomeruli at x300, and tuft tocorpuscle ratio and mean glomerular volume (GV) per biopsy werecalculated (13). Relative GV was defined as volume of an individualglomerulus divided by the mean GV from the same biopsy (4).Index of arteriolar hyalinosis was determined on serial sectionssemiquantitatively as detailed elsewhere (14). Images of renalcortex acquired by systematic uniform random sampling were usedto estimate the fractional volumes of interstitium [Vv(Int/cortex)],proximal [Vv(PT/cortex)] and distal [Vv(DT/cortex)] tubulesper cortex, and atrophic tubules per total tubules [Vv(AT/TT)]using point counting (3). Tubular profiles with short, flat,or lost epithelial cells and with thick, wrinkled, and/or duplicatedtubular basement membranes (TBM) were classified as atrophic.Serial profiles of glomeruli were examined carefully to identifyGTJ and TBCA. GTJA were classified and scored on the basis ofthe severity of tubular atrophy as previously defined (4): Shortatrophic tubules (SAT), in which the length of the atrophicsegment is a few cells (Figure 1A); long atrophic tubules (LAT),in which the atrophic segment is longer than a few cells (Figure 1B);atrophic tubules with no observable glomerular opening (ATNO),in which no connection between the Bowman’s space andthe tubular lumen is discernible (Figure 1C); and AG, in whichno tubular attachment to the glomerulus is found (Figure 1D)(4). SAT were scored + 1, LAT as +2, ATNO as +3, and AG as +4,and the index of junctional atrophy (IJA) was calculated asthe average of scores among glomeruli (4).
Figure 1. Glomerulotubular junction (GTJ) abnormalities (GTJA). (A) Glomerulus attached to a short atrophic tubule (SAT). The arrow points to the atrophic segment. (B) Glomerulus attached to a long atrophic tubule (LAT). The arrow points to the atrophic segment and tuft adhesion. (C) Glomerulus attached to an atrophic tubule with no observable opening (ATNO) and a tip lesion (arrow). (D) Atubular glomerulus (AG). *Tubular remnants that possibly belonged to the AG.
Electron Microscopy Studies.
Tissues were prepared for electron microscopy as described previously(15). Random ultrathin sections through glomeruli were stainedand examined as reported previously (15). Three nonsclerosedintact glomeruli (except in two cases in which only two suitableglomeruli were available) were used for the following estimates.Vv(Mes/glom), fractional volume of mesangial cells, [Vv(MC/glom)],and mesangial matrix [Vv(MM/glom)] per glomerulus were estimatedusing point counting (15). Sv(PGBM/glom) was estimated usinga line grid (16) at x3900 magnification. Total filtration surface(TFS) was calculated as product of Sv(PGBM/glom) and glomerularvolume. GBM width was estimated at x11,700 using the orthogonalintercept method (17).
Statistical Analyses
Statistica 5.0 (StatSoft, Inc., Tulsa, OK) was used for statisticalanalyses. AER data were logarithmically transformed. Mean groupvalues were compared by ANOVA. Homogeneity of variances wasevaluated by the Levene test. Multiple regression (forward stepwise)and piecewise linear regression analyses (PLRA) were performedto model structural–functional relationships in patientswith type 1 diabetes. The quasi-Newton method of estimationwas used to minimize loss function (maximum likelihood), andthe software was allowed to estimate one possible breakpointfor each model. All data are expressed as mean ± SD unlessspecified otherwise. P < 0.05 was considered statisticallysignificant.
Demographic and Clinical Features
Age in all groups and age at onset and duration of type 1 diabetesin the groups with diabetes were similar by design (Table 1).Hypertension was almost entirely confined to proteinuric patientswho were taking angiotensin-converting enzyme inhibitors. Therewere no group differences for HbA1c. GFR was lower in proteinuricversus NA and MA patients (Table 1).
Electron Microscopy Studies
Typical diabetic glomerulopathy changes were confirmed in thetype 1 diabetes groups (Table 2). Vv(Mes/glom) was greater inproteinuric, NA, and MA patients than in control subjects, andVv(MM/glom) was increased in proteinuric compared with NA andMA patients and control subjects. Sv(PGBM/glom) was reducedin NA and proteinuric patients compared with control subjects,whereas TFS was not different among the groups. GBM width wasincreased in NA, MA, and proteinuric patients compared withcontrol subject. GBM width in patients with type 1 diabetescorrelated directly with AER (r = 0.65, P < 0.01) and inverselywith GFR (r = –0.56, P < 0.05). Vv(Mes/glom) also correlateddirectly with AER (r = 0.67, P < 0.01) and inversely withGFR (r = –0.65, P < 0.05). No statistically significantassociations were found between Sv(PGBM/glom) or TFS and eitherAER or GFR.
Light Microscopy Studies
Mean GV was numerically increased in the groups with diabetesversus control subjects, but the difference was statisticallysignificant only between control and NA groups (Table 3). GVvariability seemed to be increased in MA and proteinuric comparedwith NA patients and control subject. Glomerular tuft to corpuscleratio was similar in all groups. Index of arteriolar hyalinosiswas increased in patients with diabetes and was significantlyhigher in proteinuric patients than in other groups. Index ofarteriolar hyalinosis directly correlated with AER (r = 0.62,P = 0.03), Vv(MM/glom) (r = 0.66, P = 0.02), GBM width (r =0.76, P = 0.004), percentage of glomeruli with ATNO (r = 0.59,P = 0.04), percentage of globally sclerosed glomeruli (r = 0.87,P < 0.00001), and Vv(Int/cortex) (r = 0.65, P = 0.02) andinversely correlated with GFR (r = –0.67, P = 0.02). Vv(Int/cortex)was increased in proteinuric compared with MA patients and controlsubjects. Vv(PT/cortex) was decreased in proteinuric comparedwith NA patients. Vv(DT/cortex) was decreased in type 1 diabetesgroups compared with control subjects and in proteinuric comparedwith NA and MA patients. Vv(AT/TT) was increased in proteinuriccompared with NA and MA patients and control subjects. AER inpatients with type 1 diabetes correlated inversely with Vv(PT/cortex)(r = –0.72, P < 0.01) and directly with Vv(AT/TT) (r= 0.72, P < 0.01). GFR in patients with type 1 diabetes correlateddirectly with Vv(PT/cortex) (r = 0.61, P < 0.05) and inverselywith Vv(Int/cortex) (r = –0.83, P < 0.0001). GTJA werepresent in only 2% of glomeruli from control subjects and NApatients and 4% of glomeruli from MA patients. AG were not foundin these groups (Figure 2). In contrast, GTJA were found in71% of glomeruli in proteinuric patients. Five (8%) of the 63glomeruli in proteinuric patients were atubular. Tip lesionswere found in all glomeruli that were attached to SAT, 77% ofglomeruli that were attached to LAT, and all glomeruli thatwere attached to ATNO (Figure 2). No tuft prolapse into proximaltubule was identified. Thickening and reduplication of Bowman’scapsules were frequent findings at the sites of adhesions, regardlessof their relation to GTJ. The space between the reduplicatedlayers of Bowman’s capsule was filled, alone or in combination,with amorphous material, foam cells, fibroblast-like cells,and capillaries (Figure 3). In several glomeruli, this spaceextended toward the GTJ and beyond, seeming to dissect TBM ofthe proximal portion of the proximal tubule (Figure 3) and tonarrow or occlude these tubular lumens. Normal proximal tubularepithelial cells were replaced by flat cells throughout thesegment with reduplicated TBM. In most instances of short segmentsof tubular atrophy, flat cells were followed abruptly by normalproximal tubular epithelial cells after the end of the dissectedsegment (Figure 4). However, in some longer atrophic segments,intermediate cells were present in this transition zone. Tubularluminal narrowing was not discernible in the subset of nephronswith short atrophic tubules without reduplicated TBM. ExtensiveBowman’s capsule reduplication was present in all AG.The glomeruli that were attached to normal tubules in patientswith type 1 diabetes were larger than in control subjects (Table 4).Glomeruli that were attached to normal tubules also were largerin proteinuric than in NA or MA patients. AG were smaller thanglomeruli that were attached to NT, SAT, or LAT, and glomerulithat were attached to ATNO were smaller than glomeruli thatwere attached to NT or LAT (Table 4). Relative GV correlatedinversely with IJA (r = –0.35, P < 0.02) and was smallerin AG than other glomeruli (P < 0.01). The tuft to corpuscleratio was not statistically significantly different among glomerulithat were attached to NT, SAT, LAT, ATNO, and AG. Globally sclerosedglomeruli were more frequent in proteinuric (16.9 ± 11.7%)compared with NA (3.4 ± 7.6%; P < 0.05) and MA (5.5± 5.0%; P < 0.05) patients and with control subjects(1.8 ± 4.0%; P < 0.01).
Figure 2. Frequency of GTJA in normoalbuminuric (NA), microalbuminuric (MA), and proteinuric (P) patients and control subjects (C). NT, normal tubules; G#, number of glomeruli.
Figure 3. A glomerulus with a tip lesion. There is an adhesion of the glomerular tuft to GTJ. The space between the reduplicated Bowman’s capsule layers (*) is filled with amorphous material. The tubular basement membrane (TBM) of the proximal portion of the proximal tubule is reduplicated (single and double arrows).
Figure 4. A glomerulus attached to an atrophic tubule. Normal tubular epithelial cells are replaced by flat cells in the atrophic segment (*). There is an abrupt transition into normal epithelial cells at the terminal point of TBM reduplication (arrowhead).
Table 4. Glomerular volume, relative glomerular volume, and tuft to corpuscle ratio of glomeruli in relation to their tubular attachmentsa
Multiple regression analyses models with Vv(Mes/glom), TFS,and GBM width as predictors explained 67% of the GFR variability(Table 5) among the patients with type 1 diabetes. The additionof the IJA to this multiple regression model increased the GFRpredictability to 75%. Glomerular structural parameters, IJA,percentage of globally sclerosed glomeruli, and Vv(Int/cortex),combined, explained 86% of the GFR variability. Vv(Mes/glom)was the only independent predictor variable in the above threemodels (P < 0.005, P < 0.002, and P < 0.01, respectively).PLRA increased the glomerular structural parameters’ explanationof GFR variability to 78% with a breakpoint at 110 ml/min. AddingIJA to the model increased the predictability of the model to86%, whereas adding both IJA and Vv(Int/cortex) increased thepredictability to 92% (Table 5). Using multiple regression analyses,Vv(Mes/glom), TFS, and GBM width explained 65% of the observedAER variability with Vv(Mes/glom) (P < 0.005) and GBM width(P < 0.05) as independent predictor variables. Incorporationof IJA increased the predictability of this multiple regressionmodel to 72% with Vv(Mes/glom) (P < 0.005) and IJA (P <0.05) as independent predictors. The addition of Vv(Int/cortex)did not basically change this model. It is interesting thatwith piecewise linear regression, glomerular structural parametersalone explained 95% of the observed AER variability (breakpointat 52 µg/min), leaving almost no room for any other variableto improve the predictions (Table 5).
This study presents two new findings in the pathology of DN:(1) GTJA are almost entirely restricted to proteinuric patients,and (2) the structural–functional relationships that bestdescribe increasing AER and decreasing GFR in patients withtype 1 diabetes are nonlinear.
GTJA are common in proteinuric patients with type 1 diabetesand are usually accompanied by TBCA at the GTJ (4). Adhesionsat this location have been called tip lesions in steroid-resistantnephritic syndrome with focal segmental glomerulosclerosis (FSGS)and tip changes in other diseases by one group of investigators(18). We have no terminology preference but use "tip lesion"here to maintain consistency with our previous publication (4).Tip lesions have been reported in a variety of proteinuric renaldiseases (5), suggesting that they are related more closelyto the severity of the glomerular permselectivity defect thanto the specific nature of the glomerular injury. However, proteinuriaoften is the clinical indication for renal biopsy, imposinga sampling bias toward more advanced disease in many glomerulopathies.This study, which included only patients who underwent researchrenal biopsies and which encompassed a wide range of the naturalhistory of DN, overcame this possible selection bias. Groupsin this study were matched for age and gender, and the type1 diabetes groups also were matched for diabetes duration, therebyeliminating these potentially confounding variables. The findingthat GTJA are restricted almost entirely to overtly proteinuricpatients with type 1 diabetes is consistent with a watershedphenomenon in which, in association with overt proteinuria,new pathologic mechanisms of injury are induced. TBCA and FSGS,although frequent in proteinuric patients, are rare in NA andMA patients despite long and similar type 1 diabetes duration.Thus FSGS is restricted to patients who have type 1 diabeteswith already established diabetic glomerulopathy. This is incontrast to experimental DN, in which in animals with glomerularhyperfiltration and capillary hypertension FSGS occurs earlyand in the absence of prominent glomerular extracellular matrixaccumulation (19,20). Thus, FSGS lesions in experimental modelsof diabetes are distinctly different in their natural historyfrom those described here, and this suggests the need for extremecaution in extrapolating from these animal models to human DN.These observations also are consistent with the natural historyof DN in type 1 diabetes. Patients who are destined to developovert proteinuria begin with a long silent period during whichAER is normal or near normal for 10 to 30 yr or longer and GFRis normal or high. Renal lesions that develop during this timemay, in fact, overlap in severity with those regularly seenin MA and in proteinuric patients, as in the present and previousstudies (3). Once persistent MA develops, lesions are, on average,worse than in NA patients, and this functional change indicatesa substantially increased risk for progression to proteinuria(2,3,21,22). However, GFR generally is preserved until proteinuriatypically ushers in a progressive GFR decline toward ESRD. Wehypothesize that a combination of podocyte and proximal tubularepithelial cell injuries in diabetes (23–25) makes theGTJ area vulnerable to synechia. Once TBCA happens, paraglomerularfiltration into and beyond Bowman’s capsule could leadto Bowman’s capsule thickening, reduplication/dissection,and periglomerular fibrosis (26–28). This aberrant filtrateflow, perhaps extending toward the GTJ and dissecting the proximalportion of the proximal TBM, could lead to GTJA, proximal tubularatrophy, and AG.
This study also demonstrated that simple linear models do notbest describe structural–functional relationships in patientswith type 1 diabetes over a wide range of AER and GFR. PLRAprovided models with substantially higher predictive values.Given the relatively small number of patients in this study,predictability could have been overestimated. Therefore, therobustness of the provided models can be determined only byadditional studies. Nevertheless, these analyses are in accordancewith the natural history of the human DN and with the hypothesisthat both GTJA and downstream tubulointerstitial injury areaccelerated by the development of high-grade glomerular permselectivitydefect. Vv(Int/cortex) was increased only in the proteinuricpatients, accompanied by a decrease from control subjects inVv(PT/cortex) and Vv(DT/cortex) and accompanied by an increasein the volume fraction of atrophic cortical tubules, consistentwith previous findings (4) and with the hypothesis that proteinuriahas noxious downstream consequences to the nephron (29,30).
All type 1 diabetes groups had glomerular enlargement, a featurenot seen with shorter diabetes durations (14,31). The glomerularurinary space, however, was not disproportionately increased,despite GTJA, which conceivably could obstruct the flow of filtratefrom Bowman’s space to the proximal tubule. This may notreflect the in vivo reality or could suggest that filtrate couldleave through paraglomerular filtration into and across Bowman’scapsule, as suggested above. Glomeruli that are attached tonormal tubules in proteinuric patients are larger than thosethat are attached to normal tubules of patients in the othertype 1 diabetes groups, perhaps representing compensatory hypertrophy.However, as described elsewhere, AG in proteinuric patientsare reduced in volume, whereas glomeruli with ATNO are of intermediatesize (4).
The findings of this study demonstrate that TBCA and GTJA areessentially restricted to patients with type 1 diabetes withovert proteinuria and, along with interstitial expansion, augmentdiabetic glomerulopathy’s explanation of GFR loss in DN.Models that allow for increased rates of GFR decline in thelater stages of the evolution of DN lesions provide a betterfit to the data than simpler linear models. New mechanisms ofnephron injury seem to be induced by overt proteinuria. It istempting to hypothesize that therapies that reduce proteinuriaalso may reduce GFR decline in DN by limiting the damaging influenceof proteinuria on the GTJ and tubulointerstitium, but this remainsto be determined.
Acknowledgments
These studies were supported by grants from the National Institutesof Health (DK13083, DK054638, and DK51975) and GCRC grant M01-RR00400.
These studies were partially presented as abstracts at the annualmeeting of the American Society of Nephrology in San Diego,CA, 2003.
We thank John Basgen and Thomas Groppoli for technical assistanceand stereologic studies on the electron microscopy specimens,Behrang Hosseini Dehkordi for digital imaging and stereologicstudies on light microscopy specimens, and Stephanie Reed forpreparing the light microscopy tissue sections. We are gratefulto Cathy Bagne and Paola Bloberger for clinical coordinationand database management.
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Abstract
Introduction
